[Show abstract][Hide abstract]ABSTRACT: GnRH neurons migrate into the hypothalamus during development. Although migratory defects may result in disordered activation of the reproductive axis and lead to delayed or absent sexual maturation, specific factors regulating GnRH neuronal migration remain largely unknown. The receptor tyrosine kinase, adhesion-related kinase (Ark) (also known as Axl, UFO, and Tyro7), has been implicated in the migration of GnRH neuronal cells. Binding of its ligand, growth arrest-specific gene 6 (Gas6), promotes cytoskeletal remodeling and migration of NLT GnRH neuronal cells via Rac and p38 MAPK. Here, we examined the Axl effectors proximal to Rac in the signaling pathway. Gas6/Axl-induced lamellipodia formation and migration were blocked after phosphatidylinositol-3-kinase (PI3K) inhibition in GnRH neuronal cells. The p85 subunit of PI3K coimmunoprecipitated with Axl and was phosphorylated in a Gas6-sensitive manner. In addition, PI3K inhibition in GnRH neuronal cells diminished Gas6-induced Rac activation. Exogenous expression of a dominant-negative form of Ras also decreased GnRH neuronal lamellipodia formation, migration, and Rac activation. PI3K inhibition blocked Ras in addition to Rac activation and migration. In contrast, pharmacological blockade of the phospholipase C gamma effectors, protein kinase C or calcium/calmodulin protein kinase II, had no effect on Gas6/Axl signaling to promote Rac activation or stimulate cytoskeletal reorganization and migration. Together, these data show that the PI3K-Ras pathway is a major mediator of Axl actions upstream of Rac to induce GnRH neuronal cell migration.

[Show abstract][Hide abstract]ABSTRACT: Gonadotropin-releasing hormone (GnRH) neurons originate in the nasal compartment, migrate along olfactory nerves to the cribriform plate and then diverge caudally to the forebrain to reach their final destination in the hypothalamus. Here, GnRH neurons are dispersed in a network that synchronously releases GnRH in a pulsatile manner to activate pituitary gonadotropin production. Gonadotropins in turn promote gametogenesis and steroidogenesis from the gonad, ensuring reproductive competence. Failure of GnRH neurons to migrate normally results in deficient activation of the reproductive axis and delayed or absent pubertal maturation. The exact mechanisms that direct GnRH neuronal migration are under active investigation. This article will review new information concerning factors regulating GnRH neuronal migration and the intracellular signaling pathways involved in this process.

[Show abstract][Hide abstract]ABSTRACT: A yeast two-hybrid assay was employed to identify androgen receptor (AR) protein partners in gonadotropin-releasing hormone neuronal cells. By using an AR deletion construct (AR-(Delta371-485)) as a bait, beta-catenin was identified as an AR-interacting protein from a gonadotropin-releasing hormone neuronal cell library. Immunolocalization of co-transfected AR and FLAG-beta-catenin demonstrated that FLAG-beta-catenin was predominantly cytoplasmic in the absence of androgen. In the presence of 5alpha-dihydrotestosterone, FLAG-beta-catenin completely co-localized to the nucleus with AR. This effect was specific to AR because liganded progesterone, glucocorticoid, or estrogen alpha receptors did not translocate FLAG-beta-catenin to the nucleus. Agonist-bound AR was required because the AR antagonists casodex and hydroxyflutamide failed to translocate beta-catenin. Time course experiments demonstrated that co-translocation occurred with similar kinetics. Nuclear co-localization was independent of the glycogen synthase kinase-3beta, p42/44 ERK mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways because inhibitors of these pathways had no effect. Transcription assays demonstrated that liganded AR repressed beta-catenin/T cell factor-responsive reporter gene activity. Conversely, co-expression of beta-catenin/T cell factor repressed AR stimulation of AR-responsive reporter gene activity. Our data suggest that liganded AR shuttles beta-catenin to the nucleus and that nuclear interaction of AR with beta-catenin may modulate transcriptional activity in androgen target tissues.

[Show abstract][Hide abstract]ABSTRACT: A yeast two-hybrid assay was employed to identify androgen receptor (AR) protein partners in gonadotropin-releasing hormone
neuronal cells. By using an AR deletion construct (AR-(Δ371–485)) as a bait, β-catenin was identified as an AR-interacting
protein from a gonadotropin-releasing hormone neuronal cell library. Immunolocalization of co-transfected AR and FLAG-β-catenin
demonstrated that FLAG-β-catenin was predominantly cytoplasmic in the absence of androgen. In the presence of 5α-dihydrotestosterone,
FLAG-β-catenin completely co-localized to the nucleus with AR. This effect was specific to AR because liganded progesterone,
glucocorticoid, or estrogen α receptors did not translocate FLAG-β-catenin to the nucleus. Agonist-bound AR was required because
the AR antagonists casodex and hydroxyflutamide failed to translocate β-catenin. Time course experiments demonstrated that
co-translocation occurred with similar kinetics. Nuclear co-localization was independent of the glycogen synthase kinase-3β,
p42/44 ERK mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways because inhibitors of these pathways
had no effect. Transcription assays demonstrated that liganded AR repressed β-catenin/T cell factor-responsive reporter gene
activity. Conversely, co-expression of β-catenin/T cell factor repressed AR stimulation of AR-responsive reporter gene activity.
Our data suggest that liganded AR shuttles β-catenin to the nucleus and that nuclear interaction of AR with β-catenin may
modulate transcriptional activity in androgen target tissues.

[Show abstract][Hide abstract]ABSTRACT: Gonadotropin-releasing hormone (GnRH) is the central regulator of the reproductive axis. Normal sexual maturation depends on the migration of GnRH neurons from the olfactory placode to the hypothalamus during development. Previously, we showed restricted expression of the membrane receptor adhesion-related kinase (Ark) in immortalized cell lines derived from migratory but not postmigratory GnRH neurons. In addition, Ark and GnRH transcripts were detected along the GnRH neuron migratory route in the E13 mouse cribriform plate. In the present study, we examined the role of Ark and its ligand, Gas6 (encoded by growth arrest-specific gene 6), in GnRH neuron migration. Gas6 stimulated lamellipodial extension, membrane ruffling, and chemotaxis of immortalized NLT GnRH neuronal cells via the Ark receptor. Gas6/Ark signaling promoted activation of the Rho family GTPase Rac, and adenoviral-mediated expression of dominant negative N17Rac abolished Gas6/Ark-induced actin cytoskeletal reorganization and migration of GnRH neuronal cells. In addition, p38 MAPK was activated downstream of Ark and Rac, and inhibition of p38 MAPK with either SB203580 or adenoviral dominant negative p38alpha also blocked Gas6/Ark-mediated migration. Finally, downstream of Rac and p38 mitogen-activated protein kinase (MAPK), Gas6/Ark signaling promoted activation of MAPK-activated protein kinase 2 and induced phosphorylation of HSP25, a known regulator of cortical actin remodeling. The data are the first to demonstrate a migratory signaling pathway downstream of Ark/Axl family receptors and suggest a previously unidentified role for p38 MAPK in neuronal migration. Furthermore, these studies support a potential role for Ark in the regulation of GnRH neuronal migration.